Engineering Escherichia coli BL21(DE3) Derivative Strains To Minimize E. coli Protein Contamination after Purification by Immobilized Metal Affinity Chromatography
Engineering Escherichia coli BL21(DE3) Derivative Strains To Minimize E. coli Protein Contamination after Purification by Immobilized Metal Affinity Chromatography
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DOI：
10.1128/aem.00119-11
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发表时间：
2011-07-01
影响因子：
4.4
通讯作者：
Samuelson, James C.
中科院分区：
文献类型：
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作者：
Robichon, Carine;Luo, Jianying;Samuelson, James C.
在大肠杆菌中表达并通过固定化金属亲和层析（IMAC）纯化的重组his标记蛋白通常与天然大肠杆菌蛋白共洗脱，特别是当重组蛋白表达水平较低时。大肠杆菌污染物对二价镍或钴离子表现出高亲和力，主要是由于存在簇状组氨酸残基或生物相关的金属结合位点。为了提高表达his标记蛋白的最终纯度，我们设计了大肠杆菌BL21（DE3）表达菌株，其中最常见的污染物要么用替代标签表达，要么突变以降低其对二价阳离子的亲和力。本研究介绍了两种大肠杆菌BL21（DE3）衍生物NiCo21（DE3）和NiCo22（DE3）的设计、工程和表征，它们表达内源性蛋白sld、Can、ArnA和AceE（可选）在其C端融合到几丁质结合域（CBD）和蛋白GlmS， 6个表面组氨酸被丙氨酸取代。我们发现，每个大肠杆菌cbd标记的蛋白都保持活性，并且可以通过几丁质柱流动步骤有效地从IMAC洗脱部分中去除，而GlmS的修饰导致对含镍树脂的亲和力丧失。“NiCo”菌株独特地补充了现有的方法，以提高重组his标记蛋白的纯度。
Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The "NiCo" strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.